Cell-based therapy for cartilage repair has gained increasing popularity since the first reports of successful autologous chondrocyte implantation (ACI) over 10 years ago (Minas et al., “Chondrocyte Implantation in the Repair of Chondral Lesions of the Knee: Economics and Quality of Life,” Am. J. Orthop. 27(11):739-44 (1998)). In ACI, primary chondrocytes are obtained from small biopsies of healthy articular cartilage, expanded, and then placed onto three-dimensional scaffolds for subsequent use in cartilage repair surgery (see Vavken et al., “Effectiveness of Autologous Chondrocyte Implantation in Cartilage Repair of the Knee: A Systematic Review of Controlled Trials,” Osteoarthritis Cartilage 18(6):857-63 (2010)). Currently, ACI is used in approximately 10% of all cartilage repair procedures performed world-wide where the lesions are less than 2-4 cm2 (Cole et al., “Outcomes After a Single-Stage Procedure for Cell-based Cartilage Repair: A Prospective Clinical Safety Trial With 2-year Follow-up,” Am. J. Sports Med. 39(6):1170-79 (2011)). ACI has also been used in veterinary medicine to improve the outcome of cartilage repair surgery in large (equine) and small (dog) animals (Breinan et al., “Autologous Chondrocyte Implantation in a Canine Model: Change in Composition of Reparative Tissue With Time,” J. Orthop. Res. 19(3):482-92 (2001); Frisbie et al., “Evaluation of Autologous Chondrocyte Transplantation Via a Collagen Membrane in Equine Articular Defects: Results at 12 and 18 Months,” Osteoarthritis Cartilage 16(6):667-79 (2008)). There have been many reports documenting the improved clinical effectiveness of ACI as compared to other cartilage repair procedures, and several large, multi-site clinical studies are currently underway (Ebert et al., “Clinical and Magnetic Resonance Imaging-based Outcomes to 5 Years After Matrix-induced Autologous Chondrocyte Implantation to Address Articular Cartilage Defects in the Knee,” Am. J. Sports Med. 39(4):753-63 (2011)). An important limitation of this procedure, however, is the requirement of two invasive surgeries, the first of which requires extraction of cells from healthy cartilage tissue, and the second to implant the cells that have been expanded ex vivo. Recent research has therefore focused on the use of alternative chondrocyte sources where the cells can be obtained less invasively (e.g., nasoseptal (Bichara et al., “Porouspoly(Vinyl Alcohol)-alginate Gel Hybrid Construct for Neocartilage Formation Using Human Nasoseptal Cells,” J. Surg. Res. 163(2):331-6 (2010))), the generation of chondrocytes from adult stem cells (e.g., mesenchymal stem cells (MSCs) from the bone marrow or adipose tissue), and/or the use of MSCs directly for transplantation (Augello et al., “Mesenchymal Stem Cells: A Perspective From In Vitro Cultures to In Vivo Migration and Niches,” Eur. Cell Mater. 20:121-33 (2010); Chanda et al., “Therapeutic Potential of Adult Bone Marrow-derived Mesenchymal Stem Cells in Diseases of the Skeleton,” J. Cell Biochem. 111(2):249-57 (2010); Hildner et al., “State of the Art and Future Perspectives of Articular Cartilage Regeneration: A Focus on Adipose-derived Stem Cells and Platelet-derived Products,” J. Tissue Eng. Regen. Med. 5(4):e36-51 (2011)).
A key factor in the development of any cell-based therapy is to find safe and effective methods to rapidly expand autologous cells in a manner that retains their phenotype and in vivo repair potential. For ACI, research has concentrated on defining the culture media and growth factors used for articular chondrocyte expansion, as well as the improved design and formulation of scaffolds used to adhere the cells and prepare them for surgical re-implantation. Currently, most culture medias used to expand primary articular chondrocytes contain serum supplemented with growth factors, including members of the transforming growth factor (TGF) (β1, β2, and β3) and bone morphogenic families (BMP) (2,4,6,12,13), insulin growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and others (see Umlauf et al., “Cartilage Biology, Pathology, and Repair,” Cell Mol. Life Sci. 67(24):4197-211 (2010)). Similarly, numerous transcription factors influence chondrogenesis, including Sox9, β-catenin, Smads, and others, resulting in optimal expression of chondrocyte-specific markers. Sox9 in particular is required for pre-cartilage condensation and differentiation of chondroprogenitor cells into chondroblasts (Lee et al., “Sox9 Function in Craniofacial Development and Disease,” Genesis 49(4):200-8 (2011)).
Evaluation of chondrocyte quality from these various cell culture procedures generally relies on documenting the expression of chondrocyte-specific markers, including various collagens (e.g., I and II), extracellular matrix components (e.g., aggregan), and growth and transcription factors known to influence chondrogenesis. Unfortunately, although many different culture systems have been used to evaluate chondrogenesis in vitro, no consensus method exists and current procedures are not very effective at maintaining the chondrogenic phenotype during the expansion period.
Similarly, many different methods have been used to expand and differentiate MSCs into chondrocytes. Since MSCs represent a very small fraction of the total bone marrow (BM) cell population, they must be enriched by techniques such as flow cytometry, or expanded in culture to obtain enough cells for transplantation. These procedures increase the risk of transformation and/or contamination of the stem cell population. In addition, following the initial expansion they must undergo an in vitro differentiation period of several additional weeks, and the chondrogenic potential of these “induced” chondrocytes remains in question (Dashtdar et al., “Preliminary Study Comparing the Use of Allogenic Chondrogenic Pre-differentiated and Undifferentiated Mesenchymal Stem Cells for the Repair of Full Thickness Articular Cartilage Defects in Rabbits,” J. Orthop. Res. 29(9):1336-42 (2011)).
The present invention is directed to overcoming these and other deficiencies in the art.